Sigma receptors (σ) have received much attention from the drug discovery field due to their possible involvement in schizophrenia, regulation of motor behavior, convulsions, anxiety, and the psychostimulant effects of drugs of abuse including cocaine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA).1,2 In addition to a host of neurological and psychiatric areas of interest, sigma receptors are promising drug development targets for, oncological, immunological, cardiovascular, opthalmological, developmental, gastrointestinal and metabolic disorders as well as those affecting the endocrine system. They are structurally unique proteins that are distinct from classical G protein-coupled receptors, ionotropic receptors, or receptor tyrosine kinases. With two subtypes currently known, they modulate cell survival and excitability, and subserve many critical functions in the body. Endogenous ligands for these receptors are unknown, though current clues point to neurosteroids.3 
The two subtypes, σ-1 and σ-2, were delineated by studies examining their respective molecular weights, distribution in tissue and drug selectivity profiles. The 223 amino acid σ-1 protein with two transmembrane spanning regions has been purified and cloned from several animal species including mouse, rat, guinea pig, and human.4-8 To date, the σ-1 receptor is well studied and known because of the receptor sequence information and availability of selective σ-1 ligands. But, the protein corresponding to σ-2 sites has not yet been cloned. Also, σ-2 receptor-selective ligands are less common, with tritiated DTG (1,3-di(2-tolyl)guanidine) being accepted as a radioligand in the presence of (+)-pentazocine (to block binding to σ-1 sites). Due to the lack of availability of detailed protein structural information and truly selective σ-2 ligands, the pharmacological characterization of the σ-2 subtype has been very limited. There is clearly a need for a selective σ-2 ligand which can not only act as a probe to explore unknown biochemical mechanisms, but also be used as a radioligand in σ-2 receptor binding assays.
The abuse of drugs is a serious social, economic and health problem worldwide. Some of the opiates, cocaine, amphetamines and phencyclidine (PCP) are the drugs of abuse with significant affinities for σ receptors. Current treatments for drugs of abuse are limited and there is a need to develop novel and effective agents to combat this problem.
Cocaine use and abuse have been reported as early as the late 1500s.9 The historical use has been associated with the chewing of leaves from the Erythroxylon coca bush, from which cocaine was isolated in 1860,10 to eliminate fatigue in workers. Indeed, cocaine is a powerful and addictive psychostimulant. Cocaine abuse is widespread and is responsible for more serious intoxications and deaths than any other illicit drug. However, the invigorating effects of cocaine have caused it to become a major recreational drug of abuse throughout the world with an estimated 13 million people using the drug. In 2004, 34.2 million Americans aged 12 and over reported lifetime use of cocaine with approximately 5.6 million reporting annual use and an estimated 2 million reporting current use of the drug. In 2004 alone, there were an estimated 1 million new users of cocaine amounting to ˜2,700 per day. Despite a decline between 2002 and 2003 which is thought to potentially be due to increases in usage of other stimulants such as methamphetamine, data from the National Survey on Drug Use and Health showed near a 70% increase in the number of people receiving treatment for cocaine addiction from 276,000 in 2003 to 466,000 in 2004.11 
Currently, there are no approved medications to treat cocaine abuse or addiction. An effective strategy used to develop an anti-cocaine agent was the development of antagonists that compete with cocaine for its target proteins. For years, treatment approaches have targeted the dopaminergic system which is known to be involved in the actions and rewards of cocaine use. Many compounds were generated and tested that targeted the dopamine transporter which was identified as a primary site of action of cocaine. These compounds were met with very limited success as many of them just substituted for cocaine.12 After many years of investigation at the dopamine transporter as well as the dopamine receptors, researchers have been challenged to envision novel mechanisms that may afford new therapeutic interventions for cocaine addiction.
Although many other mechanisms are under investigation, the a receptor system has been demonstrated and validated as a legitimate target for the attenuation of cocaine effects. The ability of cocaine to bind to the sigma receptors was discovered and first documented in 1988.13 It was reported that cocaine had a micromolar affinity for the sigma receptor, and this interaction corresponded to micromolar levels that were achievable by cocaine in the body.14 Additional studies have indicated that reducing brain sigma receptor levels with antisense oligonucleotides attenuates the convulsive and locomotor stimulant actions of cocaine. Synthetic small molecule antagonists of sigma receptors have also been shown to mitigate the actions of cocaine in animal models. From prior work, the role of the σ-1 subtype has been clearly linked to the actions of cocaine. However, the role of the σ-2 receptor has been suggested, but is less clear due to the lack of truly selective ligands for this subtype.
Radioligands selective for σ-1 receptors have the potential to non-invasively detect and monitor various pathologies, including neurodegenerative diseases and cancer.
Applicant herein reports the synthesis, radiofluorination and evaluation of a new 18F fluorinated σ-1 receptor ligands including 6-(3-fluoropropyl)-3-(2-(azapan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one (18, [18F] FTC-146). [18F] FTC-146 displays superior in vitro affinity and selectivity compared to other reported σ-1 receptor compounds. The new 18F fluorinated σ-1 receptor ligands, including [18F] FTC-146, can be synthesized by nucleophilic fluorination using an automated module. [18F] FTC-146 afforded a product with >99% radiochemical purity (RCP) and specific activity (SA) of 3.9±1.9 Ci/μmol (n=13). Cell uptake studies revealed that [18F] FTC-146 accumulation correlated with levels of σ-1 receptor protein. Furthermore, the binding profile of [18F] FTC-146 was comparable to that of known high affinity σ-1 receptor ligand (+)-[3H] pentazocine in the same cell uptake assay. PET images of [18F] FTC-146 in normal mice showed high uptake of the radioligand in the brain which is known to contain high levels of σ-1 receptors. Time activity curves (TACs) showed rapid, high initial uptake of [18F] FTC-146 in the mouse brain. Pre-treatment with non-radioactive CM304 (1 mg/kg) reduced the binding of [18F]FTC-146 in the brain at 60 min by 83% denoting that [18F] FTC-146 accumulation in mouse brain represents a specific binding to σ-1 receptors. These results indicate that [18F] FTC-146 is a good candidate radiotracer for studying σ-1 receptors in living subjects.
Initially the sigma receptor was thought to belong to the opioid class of receptors;15 however, further studies classified it as a distinct molecular entity, resulting in its recognition as a separate family of receptors.16 There are at least two σ receptor subtypes, the σ-1 and σ-2 receptors.17 The σ-1 receptor is the best characterized of the two at present.18, 19 
Despite initial controversy and conflicting ideas, recent key discoveries concerning the σ receptor have helped elucidate various biological aspects about this molecular chaperone and its putative functional roles.20,21 Mainly located at the endoplasmic reticulum of cells, σ-1 receptors have been implicated in a host of biochemical processes and pathological conditions including neurodegenerative diseases, psychiatric disorders, drug addiction, digestive function, regulation of smooth muscle contraction and ischemia.20, 22-24 σ-1 receptors are also highly expressed in most known human cancers (e.g., breast, lung, colon, ovarian, prostate, brain).24,25 Agonists for σ-1 receptors influence intracellular and extracellular Ca2+ levels and thus have a broad range of neuromodulatory effects.26,27 Certain σ-1 receptor agonists have been shown to regulate endothelial cell proliferation,28 improve cognition,29,30 provide neuroprotection,31 and act as anti-depressant agents,18,32 while antagonists inhibit/attenuate cocaine-induced seizures,33 highlighting the potential of σ-1 receptors as both a diagnostic and therapeutic target.
There are a multitude of compounds that target σ receptors, including three specific classes of compounds; 1) benzomorphans, such as (+)-pentazocine (FIG. 1) and (+)—N-allylnormetazocine (NANM) that preferentially bind σ-1 receptors (compared to their (−)-enantiomers), 2) endogenous neurosteroids like progesterone (an antagonist of the σ-1 receptor) and 3) butyrophenones, such as the antipsychotic agent haloperidol that displays high affinity for both a receptor subtypes.19,34 Over the last two decades numerous groups have reported the development of high affinity σ-1 receptor ligands34-42—and of these, some have been labeled with radioisotopes (FIG. 1) for use in positron emission tomography (PET) studies.
Examining σ-1 receptors in living subjects with PET is an important step towards understanding the receptor's functional role and involvement in disease. PET radioligands specific for σ-1 receptors could potentially provide a non-invasive means of 1) visualizing and investigating the machinery of these sites, 2) assessing receptor occupancy (to help determine optimal doses of therapeutic drugs), 3) early detection and staging of σ-1 receptor-related disease(s), and 4) monitoring therapeutic response. Some existing σ-1 receptor radioligands include: [11C] SA4503,43 [18F] FM-SA4503,44 [18F] FPS, 45 [18F] SFE,46,47-[18F] FBFPA,48 [18G] fluspidine49 and [11C]1339 (FIG. 1). The high affinity σ-1 receptor radioligand [11C] SA4503 has demonstrated promising results in rodents,43 felines50 and non-human primates,51 and is currently the only σ-1 receptor radioligand being routinely used in clinical research;52, 53 however, it is far from ideal for several reasons including its high non-specific binding, affinity for other sites such as emopamil binding protein (EBP),54 and suboptimal kinetic profile (indicative of irreversible binding). The fluorinated derivative of [11C]SA4503 (known as [18F]FM-SA4503) has demonstrated similar disadvantages in rodents and non-human primates, and is yet to be evaluated in humans. The piperidine [18F] FPS reported by Waterhouse and colleagues was evaluated in human subjects in 2003,46, 55, 56 however it displayed unfavorable kinetics (due to its inability to reach transient equilibrium at 4 h p.i.). Following these results, a lower affinity fluoromethyl derivative of [18F]FPS (known as [18F]SFE) was developed in hope of rectifying the issue of irreversible binding.46 Whilst [18F]SFE exhibited a superior kinetic profile (cleared from rat brain with a 40% reduction in peak uptake over a 90 min period), it was found to have a lower selectivity ratio, and in fact blocking studies in rats using a selective σ-2 receptor compound resulted in a small yet noticeable reduction in [18F]SFE uptake.46 In 2005 Mach and colleagues reported the radiosynthesis of another piperidine derivative [18F]FBFPA (affinity for σ-2 receptor/σ-1 receptor=44) and demonstrated its ability to bind σ-1 receptors in both rodent and rhesus monkey brain.48 In 2010 the synthesis of a spirocyclic piperidine σ-1 receptor radioligand, [18F]fluspidine, and its evaluation in mice was reported.37, 49 Biodistribution results showed 40% reduction in brain [18F]fluspidine uptake over 2 hours, indicating that it may display reversible binding; however, it is still in the early stages of evaluation. Moussa and colleagues published the radiosynthesis of a carbon-11 labeled N-benzyl piperazine σ-1 receptor ligand, [11C] 13, and its in vivo evaluation in Papio hamadryas baboons using PET imaging. Whilst [11C] 13 accumulated in sigma-1 rich regions of the brain and peripheral organs, it was found to display a low selectivity ratio (affinity for σ-2 receptor/σ-1 receptor=38) and also a nanomolar affinity for 5-HT2B receptors.39 
Until the present patent application, there was no highly selective σ-1 receptor radioligand labeled with fluorine-18 or carbon-11 available for clinical research.
Alzheimer's Disease (AD) is a progressive degenerative brain disorder that destroys brain cells, causing memory loss and problems with thinking and behavior severe enough to affect work, lifestyles, or social life. Sigma-1 receptors (S1Rs) have been shown to be critical target in the treatment of memory deficits and cognitive disorders including AD. S1R is implicated in cellular differentiation [37,40], neuroplasticity [145,149], neuroprotection [71,89], and cognitive functioning of the brain [85] [Waarde Reference]. Previous studies showed a decrease of sigma receptor density in aging and neurodegenerative disease by autoradiography in monkeys (e.g., [3H] DTG) and positron emission tomography (PET) in human (e.g., [11C] SA4503).
PET imaging of S1Rs has the potential to non-invasively detect and monitor the numerous pathologies in which this receptor plays a role, building upon the established ability of PET to quantify specific ligand-receptor binding in the brain.” Although several S1R-binding compounds 3-10 have been made, [11C] SA4503 is currently the only radiotracer used for imaging S1R in the clinic, 11 despite its moderate selectivity for other targets including the sigma-2 receptor. Thus, the goal of this proposal is to develop and apply a more selective PET imaging S1R-selective ligand as a biomarker for therapeutic drug discovery and for the pathophysiological study of Alzheimer's disease.
It is an object of the present invention to develop a highly selective novel ligand or radioligand to image the action of these proteins in vivo in order to facilitate the understanding of various biological aspects about this molecular chaperone and its putative functional roles, and to accelerate the design and evaluation of novel molecular targeted therapies against AD.
Thus, the goal of this proposal is to develop and apply a more selective PET imaging S1R-selective ligand as a biomarker for therapeutic drug discovery and for the pathophysiological study of Alzheimer's disease.
Peripheral nerve injury, as a consequence of trauma, surgery, inflammation, degenerative changes, diabetes, and a variety of other causes, is a major clinical problem resulting in significant morbidity such as chronic pain, weakness, and other sensorimotor disabilities. Consequently, peripheral nerve injury and neuroinflammation are an overwhelming public health problem, and often require significant resources for the diagnosis and treatment of patients with chronic pain, nerve regeneration, and other related conditions.
Current methods to diagnose nerve injury include computed tomography (CT), ultrasound imaging (US), magnetic resonance imaging (MRI) and electrophysiologic (EP) (i.e., Electrodiagnostic or electroneurography) tests, namely, electromyography, quantitative neurosensory testing, and nerve conduction studies. In particular, the EP tests can be helpful in identifying conduction abnormalities and grading the extent of nerve injury in the interrogated regions, but the results of these studies are susceptible to a variety of limitations. For example, EP tests are invasive often requiring multiple passes of the needle in regions of interest to derive a diagnosis. Additionally, the results of these tests provide limited information about the cause and the location of the injury and are temporally-dependent relative to the timing and extent of nerve injury. EP results are also open to technical and operator-dependent errors, including the interpretation of the waveform results, which is a relatively subjective experience that can potentially lead to inaccurate conclusions (77).
By comparison, currently employed clinical imaging methods used to diagnose peripheral nerve injury, such as MRI, may be able to provide better insight as to the cause and the location of the nerve injury itself and secondary consequences of muscle denervation (78). However, the correlation between MRI and EP tests in detecting such lesions remains suboptimal. For example, investigators have found that only half of those individuals presenting with carpal tunnel syndrome with confirmed electrophysiologic abnormalities of the median nerve show an abnormality on MRI (79). Others have also found no correlation between EP studies and MR findings of the peripheral nerve (80) and in some cases there are no specific EP findings or imaging findings in certain patients (80, 81).
Even the challenges of current clinical methods, the identification of molecular imaging approaches that exploit molecular markers of nerve injury or neuroinflammation, and thus highlight the location and extent of nerve injury, is of paramount importance to advancing the management of nerve injury, neuroinflammation, and the ensuing clinical manifestations of these entities. While MRI has unparalleled soft tissue contrast and ultra-high spatial resolution, it suffers from poor sensitivity, and is limited in terms of its currently available clinical molecular imaging applications. Positron emission tomography (PET) is a molecular imaging technique, which is ideally suited for monitoring cellular and biochemical events early in the course of a disease due to its high sensitivity, unlimited depth of penetration, non-invasive nature, and quantitative capabilities. The combination of PET with MRI is an exciting prospect; as one can leverage the advantages of each imaging technique—i.e., high sensitivity and spatial resolution—to simultaneously visualize biochemical and anatomical alterations. While the use of PET-MRI has not yet been reported for clinical imaging of chronic pain and/or nerve injury, it holds great promise for improving the way we identify regions of nerve injury and pain generators, and thus the diagnosis and treatment of chronic pain and related conditions.
A potential biomarker associated with nerve injury and neuroinflammation is the sigma-1 receptor (S1R), which was initially believed to be a subtype of opioid receptor (15), but is now known to be a distinct class of receptors with unique biological functions (20,18, 83). S1R antagonists, for example, are known to modulate opioid analgesia (84), and drugs such as haloperidol, which bind S1Rs, can augment the anti-nociceptive effect of opioids (85). In addition, S1Rs can modulate various ion channels and receptors, including potassium channels, calcium channels, dopamine and gamma-amino butyric acid (GABA) receptors (86-88), thereby significantly impacting neural excitability and transmission by affecting the release of several neurotransmitters including serotonin, dopamine, noradrenaline, glutamate, and GABA.
With respect to pain, it has been known for quite some time that S1R agonists inhibit opioid analgesia, whereas antagonists enhance analgesic effects (84, 90). Furthermore, S1R knockout mice showed decreased response to pain in various pain models (31, 60, 92). Treatment with S1R antagonists such as haloperidol and its metabolites I and II also produces similar results (93, 94). Further, spinal S1R activation can result in mechanical and thermal hypersensitivity (95) and increased N-methyl-D-Aspartate (NMDA) receptor-induced pain (96, 97) while spinal S1R inhibition alleviates pain behavior (60, 94, 98). S1R is involved in synaptic plasticity and central sensitization, which are implicated in the “memorizing” of pain responsible for making it chronic and self-perpetuating (60, 92). It is not surprising that S1R antagonists are quickly becoming popular as potential candidates for the next generation analgesics (99). BD1047 is a selective S1R antagonist with high affinity that has recently been successfully tested as an analgesic in animal neuropathic pain models (100).
Since S1Rs are involved in nociception, it would be extremely valuable to have a tool which could help us better understand the role of these receptors in vivo in pain/nerve injury, potentially leading to better approaches to diagnose and treat pain. Applicant has recently developed a highly selective radiotracer, [18F] FTC-146, for imaging S1Rs with PET, and have demonstrated its specificity in mice, rats, and monkeys (Scheme 1) [ref, James et al submitted, JNM]. Here applicant aims to employ [18F]FTC-146 as a tool for visualizing S1Rs in a rat model of nerve injury so that applicant might gain information about S1R levels during nerve injury and whether the S1R might be a useful in vivo imaging biomarker of nerve injury.